1. Field of the Invention
This invention relates to the biological industry (medical and veterinary), and more particularly, it relates to a method of preparing attenuated viral preparations that retain specific antigen properties, useful in prophylaxis or laboratory diagnosis. Still more particularly, this invention relates to a method of attenuating the infectiousness of viruses with a simultaneous stabilization of their antigens.
2. Description of the Prior Art
It is known that specific antigenic properties of viruses are connected with protein macromolecules of viral membranes, while the infectiousness (that is, the infecting power) depends on a nucleic acid which is present at the center of the virus. It is known also that for preparing attenuated (devoid of infectious activity) viral preparations, suitable, for example for prophylactic vaccination against viral diseases or for safe use in diagnostic serological reactions, it is necessary to destroy the structure of the nucleic acid of the virus, and at the same time to protect viral proteins, the carriers of specific antigenic and immunogenic properties, against undue denaturation and to stabilize them as well. The processes of attenuating infectiousness and stabilizing antigen viral proteins should take place simultaneously.
When placed in a suspension of cells of affected tissues, or cell-free substrate, pathogenic viruses, for example, the causative agents of encephalitis and hemorrhagic fever, are usually destroyed by themselves at temperatures above the water freezing point. The rate of inactivation is different in various viruses (with consideration of the protective action of some substrates containing the viruses), but in general, it is proportional to the elevation of the temperature. The loss of infection power under the action of temperatures, is, as a rule, accompanied by a progressive decrease in the antigenic properties of the preparation and the complete loss of same. In this connection, methods of attenuation of infectiousness of viruses, which make it possible at the same time to protect their antigens from excess reduction in activity with increasing temperature, are used. These methods stabilize virus protein antigens and prevent their destruction. For example, known in the prior art are methods of chemical treatment of a viral preparation with phenols, beta-propiolactone, and formaldehyde, in combination with a more or less short duration of temperature on the order of 37.degree., 32.degree., 24.degree., and 4.degree.C, depending on the nature of the virus (cf. Gard, S., Chemical Inactivation, "Nature of Virus," Moscow, 1958).
The process of virus inactivation should be carried out under conditions which completely destroy virus infectiousness with preservation of their maximum antigenic properties none of which can be attained with the above-named chemical substances.
For example, formaldehyde is most often used as an inactivator of viruses in the manufacture of vaccines against viral encephalitis and hemorrhagic fever. It is used in a concentration from 2.4.times.10.sup..sup.-2 to 6.times.10.sup..sup.-3 M, depending on the substrate containing the virus. Its optimum dose is difficult to select and this results in excess denaturation of antigen proteins and reduction of the final preparation's immunogenicity. When treated with formaldehyde in said concentrations, the antigens used for the complement fixation test, become anticomplementary. Therefore, they cannot be used in the test.
Another disadvantage of using formaldehyde as an inactivating agent concerns the length of the inactivation process which is detrimental to the immunogenic activity of vaccine.
In order to compensate for the inevitable loss of antigenic activity of the viral preparation due to the destructive effect of formaldehyde, the concentration of the virus in the material should be very high, which is difficult to attain under conditions of an industrial enterprise. Moreover, the presence of free formaldehyde in the vaccine produces pain in those vaccinated; accordingly special deformalinization of vaccine is necessary.